We have previously found that docosahexaenoic acid (DHA, 22:6n-3), a highly polyunsaturated n-3 fatty acid enriched in neuronal tissues, promotes the accumulation of phosphatidylserine (PS) and prevents apoptotic cell death in a PS- and PI3 kinase-dependent manner in neuronal cells. We have also demonstrated that n-3 fatty acid deficiency or chronic ethanol exposure markedly decreased the PS content specifically in neuronal cells, adversely affecting neuronal survival. We have also established that DHA promotes neurite outgrowth in hippocampal neurons, suggesting a role of DHA in neuronal differentiation. During this period, we continued our investigation on the signaling mechanisms underlying effects of DHA and ethanol on neuronal survival and development. We also continued to evaluate the role of DHA in development of hippocampal neurons. [unreadable] Analysis by immunocytochemistry indicated that DHA supplementation stimulated both dendrite growth and synapse formation in hippocampal neurons. The synapsin staining in control cells after 14 days in vitro is continuous throughout the axons, while DHA supplemented cells showed a discontinuous distribution of synapsin staining as puncta-like spots which are considered to represent synapse formation. Synapse formation at 14 DIV quantified by the number of synapsin positive dots in neurons showed that DHA significantly increased the total number of synapsis per area in comparison to the control. The effect of other fatty acids such as oleic and arachidonic are currently being evaluated. [unreadable] As we found that Akt was a target molecule for the DHA?s antiapoptotic effect, we examined Akt conformational changes during activation using chemical cross-linking and tandem mass spectrometry. Our cross-linking data revealed a novel Akt activation scheme where Akt undergoes stepwise inter-domain conformational changes in the sequential activation stages including membrane interaction, phosphorylation and substrate binding. During this report period, we focused on membrane-Akt interaction, especially the effect of membrane composition on Akt conformational changes and activation. Mass spectrometric analysis revealed that the presence of both PIP3 and PS in the membrane is required for Akt conformational changes needed for phosphorylation for its full activation. Consistently, in vitro Akt-phosphorylation at T308 and S473, by PDK and MAPKAP, respectively, is dependent on concentrations of both PIP3 and PS in the membrane. Moreover, PS dose-dependently enhanced the membrane interaction and phosphorylation of Akt at a given concentration of PIP3, suggesting a role of PS in complementing PIP3. Both mass spectrometric and biomolecular interaction analysis indicated that not only the PH domain but also the regulatory domain of Akt interacts with PS or PIP3. Membrane translocation of the GFP-tagged regulatory domain in response to IGF stimulation also supported the occurrence of regulatory domain-membrane interaction during Akt activation. The regulatory domain-membrane interaction was affected more prominently by PS in comparison to PIP3. In fact, the regulatory domain interacted with membranes containing PS but no PIP3 and enabled phosphorylation of S473 in the regulatory domain in vitro by MAPKAP. Our data represent the first demonstration of the distinctive molecular interaction between individual Akt domains and acidic phospholipids in cell membranes. The role of PS complementing PIP3 in Akt activation may be important in neuronal cell survival, particularly under adverse conditions where PIP3 production is compromised. This mechanism may provide an explanation for the PS-dependent neuronal survival affected by the DHA status and ethanol observed in our study.